The transfer of therapeutic genes into hematopoietic stem cells (HSC) offers the potential for effective and permanent treatment of a wide spectrum of gene-based diseases including inherited metabolic diseases, viral infections, and cancer. The hematopoietic system is comprised of a hierarchy of cells with different capacities to regenerate all the cells of the blood and bone marrow. Transfer of genes into the most primitive cells (i.e., those most capable of reconstituting the hematopoietic system) is important to the success of genetic therapies. While many studies have evaluated gene transfer into CD34 cells, transduction of stem cells which reside in a quiescent, mitotically dormant, highly primitive state has been difficult to achieve. Despite promising results in vitro, and in murine models, the results of retroviral human gene therapy trials to date have revealed disappointingly low gene transfer frequencies into pluripotent long term repopulating cells. While lentivirus vectors may offer one alternative, their inability to integrate in quiescent cells in addition to safety issues make it imperative to develop other approaches of stable gene transfer into this important cell population.
Despite the widespread use of retrovirus vectors in the majority of human gene therapy trials currently underway, several limitations remain. Human trials and nonhuman primate models of hematopoietic progenitor cell transplantation suggest that the efficiency of retroviral gene transfer into hematopoietic cells is quite low. Results of clinical gene therapy trials utilizing transplantation of retrovirus vector-transduced CD34 cells derived from either bone marrow, cord blood or mobilized peripheral blood, have been informative in determining the potentials of retroviral transduction of human hematopoietic stem cells in vivo. The majority of the results suggest a long term marking frequency of only approximately 1:10,000-1:100,000.
High retroviral transduction frequencies of hematopoietic progenitor cells in vitro, with colony forming unit (CFU) assays and long term culture initiating cell (LTC-IC) assays have been reported. But while gene transfer with retrovirus vectors in lineage-committed CFU have been successful, these studies have not been predictive of in vivo results. Moreover, murine studies showing efficient retroviral transduction of long term hematopoietic repopulating cells did not correlate with large animal models and human gene therapy trials. This discrepancy between the levels of retroviral transduction of human hematopoietic cells in vitro and in vivo is likely attributable to the cell cycle status of the cells at the time of transduction.
Retrovirus vectors pseudotyped with the VSV G protein were found to infect but not integrate into CD34+38− cells shown to exist in a nondividing state for at least 72 hours in culture. This suggests that a CD mitotic block rendered these cells untransducable by retroviruses. CD34+38− cells which gave rise to CFU only after 60-100 days in culture on stromal layers in extended LTC-IC assays were found to be both quiescent and refractory to retroviral transduction.
Transplantation of retrovirus transduced human CD34 cells in beige/nude/xid (bnx) mice resulted in the detection of common integration patterns in T lymphoid and myeloid lineages, suggesting transduction of a common progenitor. However, the frequency was again very low, with only three common T and myeloid integrants being detected from 24 mice, each transplanted with 106 transduced cells. In addition, prolonged cytokine stimulation of originally cytokine non-responsive primitive progenitor cells was necessary for retroviral transduction.
Non-lentiviral retroviruses require mitosis-induced nuclear membrane breakdown for entry into the nucleus and subsequent viral integration. Primitive hematopoietic progenitor cells induced into cell division by cytokine stimulation are transducable with retrovirus vectors, as are the majority of LTC-ICs, however there is much evidence to suggest that pluripotent hematopoietic stem cells which are required for long term multilineage reconstitution of the hematopoietic system reside primarily in the G0 phase of the cell cycle, with only a few clones dividing at any given time.
Hematopoietic stem cells may be induced to initiate mitosis by exposure to cytokines, but cytokines also induce differentiation. Thus, using this method, the resultant genetically modified cells are no longer pluripotent cells capable of self-renewal and multilineage differentiation. Recent data also suggest that other factors such as a paucity of retrovirus receptors and blocks to reverse transcription may play additional roles in the inability of retroviruses to transduce primitive hematopoietic progenitor cells. Thus, the continued search for other vectors for the safe, stable and efficient introduction of transgenes into hematopoietic stem cells is imperative.
Pluripotent human hematopoietic stem cells with the capacity to self-renew and give rise to progeny of all hematopoietic lineages, including lymphoid, myeloid and erythroid cells, may be purified from either cord blood, bone marrow or mobilized peripheral blood. Immunophenotypic analysis reveals that hematopoietic stem cells are contained within the CD34+ compartment of hematopoietic cells. Further subdivision reveals that pluripotent human hematopoietic stem cells are CD38−, lack lineage-specific differentiation markers and exclude Rhodamine 123. Cells possessing these characteristics are very primitive, highly pluripotent, mitotically quiescent and maintain a minimal level of metabolic activity.
Recent studies defined non-dividing CD34 cells as G0 on the basis of their low RNA and DNA content. Gothot t al., Blood 90(11):4384-4393 (1997); Ladd e al., Blood 90(2):658-668 (1997); Gothot et al., Exp. Hematol. 26(7):562-570 (1998); Donnelly et al., Exp. Hematol. 27(5):788-796 (1999). Some of these G0 CD34 cells have been shown to remain cytokine nonresponsive for at least seven days in culture with cytokines and to be enriched in extended long-term culture-initiating cell assays (LTC-IC). LTC-IC assays are well-known in the art. However, culture conditions that allow prolonged hematopoietic stem cells survival and self-renewal are as yet undefined. As discussed above, the cytokines that normally are added to stem and progenitor cell cultures to maintain viability, also induce undesired lineage commitment and differentiation.
The lack of appropriate direct assay systems to test self-renewing pluripotential human stem cells in vitro also has impeded attempts to analyze gene transfer into this population. Short term CFU assays test the ability of progenitor cells to divide and differentiate into lineage-specific colonies and primarily measure committed late-stage progenitors but not stem cells. Long term marrow stroma cultures with a hematopoietic microenvironment capable of supporting hematopoiesis for five weeks or longer have been described. Southerland et al., Blood 74:1563 (1989). In these cultures, more primitive colony forming cells (CFC) can survive five weeks or longer and still retain the capacity to differentiate late in culture.
While LTC-IC bear some correlation to long-term repopulating cells in animal models and are enriched in immunophenotypic compartments which are also enriched for stem cells, gene marking studies indicate that LTC-IC do not represent the pluripotent hematopoietic stem cells capable of long term lympho-myeloid reconstitution. However, this assay is still used extensively to measure primitive myelo-erythroid progenitor activity.
Extended LTC-IC have been described which measure the ability of primitive CD34+38− cells to give rise to CFCs after 60 days of culture on a radiated stroma layer. Hao et al., Blood 88:3306-3313 (1996). These cells have been shown to possess mitotic and immunophenotypic characteristics of very primitive progenitor cells.
Attempts also have been made to test human hematopoiesis in immunodeficient mice. Cell populations containing hematopoietic stem cells have been transplanted into mice bearing the severe combined immuno deficiency (SCID) mutation (rag2−/−) or beige/nude/xid (bnx) mutations. SCID mice lack mature T or B cells but have elevated levels of natural killer (NK) cells and serum complement and with age develop leakiness in the B cell compartment. While engraftment of human hematopoietic cells have been observed in SCID mice that have been provided with either human bone/thymus/liver grafts or human cytokines, the levels of engraftment are low. It is postulated that in SCID mice the elevated levels of NK cells are responsible for natural resistance to marrow grafts and the presence of serum complement contribute to low levels of human engraftment. Bnx mice, which lack functional B, T and NK cells, show somewhat improved levels of engraftment with these cells.
Recently, the SCID mutation was back-crossed onto the non-obese diabetes (NOD/Lt) background (NOD/SCID). Shultz et al., J. Immunol. 154:180 (1995). These mice have multiple defects in adaptive and innate immunity with no functional mature T or B lymphocytes, no NK cells, no complement, and defective macrophage function. They support engraftment of human hematopoietic cells better than SCID or bnx mice. Human CD34 cells engrafted in NOD/SCID mice home to the bone marrow where B lymphopoiesis, myelopoiesis, and erythropoiesis occur. Human hematopoiesis in the NOD/SCID mice is accompanied by the continued production of self-renewing CD34+ progenitors, colony-forming cells (CFC) and cells capable of giving rise to hematopoiesis upon serial transplantation. CFC present in the marrow of human engrafted NOD/SCID mice up to is ten weeks post transplantation were shown by thymidine suicide studies to be derived from quiescent cells. Therefore, NOD/SCID mice provide a model for the analysis of gene transfer into quiescent human hematopoietic stem/progenitor cells and a better predictor of successful genetic therapy for human disease.
Lentivirus vectors have recently gained attention for their ability to transduce non-dividing cells and pseudotyped lentivirus vectors have been shown to infect CD34+ cells. However, the majority of the transduced non-dividing cells were either in G1 or G2. A recent study reports that despite evidence of entry of certain lentivirus vectors into non-dividing CD34+38− cells, stable integration and transgene expression could not be detected. Therefore, despite the ability of lentiviruses to enter the nuclei of non-dividing cells, other factors may pose significant impediments to gene transfer into stem cells in the G0 phase of the cell cycle using these types of vectors. Additionally, there are significant safety concerns regarding the use of lentivirus vectors in clinical trials, especially for diseases other than HIV infection.
For stem cell gene therapy to become an effective therapeutic modality, higher levels of gene transfer in hematopoietic stem cells must be attained. AAV vectors are attractive vehicles since they (1) transduce CD34+ and CD34+38− cells, (2) transduce non-dividing cells, (3) show chromosomal integration, (4) do not encode viral genes and are not highly immunogenic, and (5) are non-pathogenic.
Adeno-associated virus (AAV, a parvovirus), is a single-stranded replication-defective DNA virus with a 4.7 kb genome having palindromic inverted terminal repeats (ITR) which are important for viral integration. Co-infection with a helper virus, typically adenovirus or herpes simplex virus, is required for productive infection. In the absence of helper virus co-infection, AAV stably and efficiently integrates via the ITRs into cellular DNA. DNA to be transferred using AAV must be contained within the AAV ITRs. Latent wild-type AAV infections have been stably maintained in tissue culture for greater than 100 serial passages in the absence of selective pressure, attesting to the stability of AAV genomic integration. AAV vectors have demonstrated high transduction frequencies in cells from diverse species (chickens to primates) and in lineages including those of hematopoietic origin.
Other advantages to AAV transduction include the fact that DNA polymerase, the enzyme responsible for AAV replication, has increased fidelity as compared to reverse transcriptase, which has a 100-10,000 fold higher error frequency. In addition, wild-type AAV has yet to be definitively identified as a pathogen in either animals or humans. On the contrary, there is evidence that infection with wild-type AAV inhibits transformation by papilloma viruses and activated H-ras oncogene in vitro. In addition, epidemiological studies suggest that prior infections in humans may confer oncoprotection. Moreover, AAV vectors which do not encode viral genes have been shown to have low immunogenicity, and prolonged in vivo transgene expression from AAV vectors has been documented.
The use of an AAV vector to deliver an antisense gene targeting the RNA sequences present in the 5′- and 3′-regions of HIV-1 mRNA has been described (Chatterjee et al., Science 258:1485-1488 (1992). Transduced cells showed specific and significant inhibition of HIV LTR-directed gene expression and virus replication. AAV transduction was not associated with any toxicity or alterations of cell viability, growth inhibition or heterologous transcription. This study represented the first use of an AAV-based anti-HIV vector. See also U.S. Pat. No. 5,474,935, which describes AAV vectors based on CWRSV and their use to transfer genes to bone marrow cells.
AAV vectors encoding ribozymes to HIV and viral-transforming genes have been developed. AAV vectors have been used to express the human tyrosine hydroxylase II gene, factor IX, neuropeptide Y, human glucocerebrosidase and arylsulfatase A, the CFTR gene, beta globin and antisense to alpha globin. Studies have been published showing AAV transduction of primary non-dividing cells, for example, post-mitotic neurons in vivo, olfactory tubercle, piriform cortex and multipolar neurons, cochlear cells, retinal cells, glial cells of the human central nervous system, non-proliferating respiratory epithelial cells, alveolar stem cells, adult skeletal muscle, cardiac muscle, noncycling tumor cells and primary human peripheral blood monocyte-macrophages.
It previously has been shown that AAV vectors can transduce non-dividing cells, for example smooth muscle cells and neurons, both in vivo and in vitro. There also have been recent demonstrations of the capacity of AAV vectors to transduce clonogenic CD34+ and CD34+/CD38− human hematopoietic progenitor cells and to display chromosomal integration. Fisher-Adams et al., Blood 88:492 (1996); Chatterjee et al., Blood 92:1882 (1999). Successful genetic transformation of extremely primitive CD34+++/CD38− cells which demonstrably are in the G0 phase of the cell cycle has not previously been shown.
The capsid proteins comprising AAV virions possess nuclear localization signals, which facilitate their entry through the nuclear pores of non-dividing cells. Wild type AAV is unique among eukaryotic viruses in its ability to integrate site-specifically into the AAVS 1 site of the human chromosome 19. This integration is mediated by the virus-encoded rep78 protein which recognizes consensus sequences on both the AAV LTR and AAVS1. Rep78 possesses site specific, DNA-binding, endonuclease and helicase activities and is postulated to form a bridge between the wild type AAV genome and AAVS1 to facilitate site-specific integration. Rep-free, wild type-free AAV vectors, however, do not integrate into AAVS1.
Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. The ability to insert transgenes into pluripotent hematopoietic stem cells residing in the G0 phase would represent a significant advance in the field of human gene therapy since the ability to deliver genes to the appropriate population of cells has been a major stumbling block until the present time.
AAV vectors have recently been approved for use in clinical gene therapy for cystic fibrosis based on recent observations of long-term in vivo expression of an AAV vector-encoded cystic fibrosis transmembrance conductance regulatory gene in rabbit airway epithelial cells. For clinical applications, the ability to transfer genes efficiently into a large fraction of hematopoietic progenitor cells in the absence of drug selection is highly desirable because the long-term effects of drug selection on the fate of transduced cells in vivo is unknown. There has been no prior disclosure of the transduction of CD34+ cells that were highly primitive, non-dividing cells residing in G0, therefore a significant need remains in the art for methods which can achieve this.